Preterm birth is a major public health concern defined as delivery prior to 37 completed weeks of gestation. Each year, up to 360,000 pregnancies deliver preterm. Preterm infants are at greater risk of death and disability than are term infants. Despite decades of research, primary prevention strategies to reduce or eliminate spontaneous preterm birth (sPTB) are lacking. The proposed study will explore the role of fetal hypothalamic-pituitary-adrenal (HPA) axis activation in sPTB and identify potential new therapeutic targets for primary sPTB prevention. The objective of this proposal is to determine whether sPTB is associated with differential fetal HPA axis gene expression compared to term births. Our central hypothesis is that fetuses that are delivered preterm have more up-regulation of HPA axis gene expression compared to those that are delivered at term. We formulated our central hypothesis based on published literature showing that corticotrophin-releasing hormone (CRH), a hypothalamic neuropeptide that regulates HPA axis activity, is integral to the regulation of labor. We will test our central hypothesis by completing two specific aims: 1: To quantify second trimester fetal expression of the key regulatory genes of the HPA axis using previously collected second trimester amniotic fluid specimens in spontaneous preterm and term births; 2) To prospectively measure fetal HPA axis-associated gene expression in both the fetal (amniotic fluid and umbilical cord blood) and maternal (blood) compartments obtained at the time of delivery of spontaneously preterm and term infants. These aims will be achieved through a retrospective and prospective analysis of amniotic fluid supernatant from women with sPTB (<34 weeks) or spontaneous term birth (>37 weeks). Fetal HPA axis gene expression will be quantified via real- time polymerase chain reaction (RT-PCR) after extraction of cell-free fetal RNA. In addition, whole genome microarray expression arrays will be used to identify other potential fetal pathways up or down-regulated in sPTB. Fetal biomarkers related to sPTB will be identified in the prospectively collected maternal blood by RT-PCR. The approach is innovative because our proposal is the first to use amniotic fluid supernatant in previously collected and prospectively collected deliveries to isolate cffRNA and measure fetal HPA axis-associated gene expression in sPTB and term birth. The proposed research is significant because identifying differential gene expression of the fetal HPA axis among pregnancies complicated by sPTB will allow us to develop new strategies to test for primary sPTB prevention. Ultimately, such knowledge has the potential to inform novel therapies to prevent sPTB.